How cells are committed to terminal differentiation is a question of basic biological importance and, for the many tissues that are continuously renewed throughout adult life, this question is central to an understanding of the maintenance of tissue homeostasis and pathology. Previous work indicates that the progenitor cells of stratifying epithelia are not equipotential and predicts a subpopulation of less-differentiated, slowly- cycling stem cells on which maintenance of tissue structure is ultimately dependent. The majority of proliferative cells are predicted to be restricted to differentiation and amplification divisions. The objectives of the studies now proposed are to use newly-developed techniques to test hypotheses of explanatory and functional interest to this concept A) That stem and amplifying cells differ in their regenerative abilities. Subpopulations of the basal cell population will be examined, using assays of cell proliferation and known differentiation markers, to determine their patterns of in vitro clonogenicity and of entry into terminal differentiation. Viable subpopulations of dissociated epithelial cells will be isolated by fluorescence activated cell sorting using monoclonal antibodies raised against cell surface epitopes expressed early in differentiation. Work will also continue to define population structure by using monoclonal antibodies to new markers and by examination of transgenic mice carrying an early differentiation marker. B) That stem and amplifying cells differ in their responses to growth factors. The differential effects of certain growth factors that are known to influence epithelial proliferation or differentiation will be assayed in vitro. The patterns of expression of relevant growth factor receptors of basal cell subpopulations will be determined. C) That epithelia consist of a series of functional units of structure corresponding to stem cell territories The basic hypothesis predicts a lineage of cells derived from each functional stem cell but the in vivo distribution of functional stem cells and the size of the units of differentiating cells that each stem cell generates are unknown. A defective recombinant retrovirus will be used to introduce a histochemically-detectable, heritable marker into a fraction of the stem cell population to determine in vivo cell lineage.